Nitric oxide regulates homeoprotein OTX1 and OTX2 expression in the rat myenteric plexus after intestinal ischemia-reperfusion injury.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

Antibiotic treatment-induced dysbiosis differently affects BDNF and TrkB expression in the brain and in the gut of juvenile mice.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

Effects of chronic desipramine treatment on alpha2-adrenoceptors and mu-opioid receptors in the guinea pig cortex and hippocampus.

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

Changes in hyaluronan deposition in the rat myenteric plexus after experimentally-induced colitis.

Myenteric plexus alterations hamper gastrointestinal motor function during intestinal inflammation. Hyaluronan (HA), an extracellular matrix glycosaminoglycan involved in inflammatory responses, may play a role in this process. In the colon of control rats, HA-binding protein (HABP), was detected in myenteric neuron soma, perineuronal space and ganglia surfaces. Prominent hyaluronan synthase 2 (HAS2) staining was found in myenteric neuron cytoplasm, suggesting that myenteric neurons produce HA. In the myenteric plexus of rats with 2, 4-dinitrobenzene sulfonic (DNBS)-induced colitis HABP staining was altered in the perineuronal space, while both HABP staining and HA levels increased in the muscularis propria. HAS2 immunopositive myenteric neurons and HAS2 mRNA and protein levels also increased. Overall, these observations suggest that inflammation alters HA distribution and levels in the gut neuromuscular compartment. Such changes may contribute to alterations in the myenteric plexus.

Postnatal development of P2 receptors in the murine gastrointestinal tract.

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADP>or=alpha,beta-meATP>or=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.

Involvement of glutamate receptors of the NMDA type in the modulation of acetylcholine and glutamate overflow from the guinea pig ileum during in vitro hypoxia and hypoglycaemia.

The involvement of NMDA glutamate receptors in the effects of glucose/oxygen deprivation (in vitro ischaemia) on spontaneous endogenous acetylcholine and glutamate overflow from the guinea pig ileum was studied. Neurotransmitter overflow was measured by HPLC. Deprivation of glucose in the medium slightly reduced acetylcholine overflow, and did not significantly influence glutamate overflow. During oxygen deprivation and glucose/oxygen deprivation, acetylcholine overflow augmented with a biphasic modality: an early peak was followed by a long lasting increase, whereas glutamate overflow increased with a rapid and sustained modality. The effects of glucose/oxygen deprivation on both acetylcholine and glutamate overflow were abolished after reperfusion with normal oxygenated medium. Acetylcholine and glutamate overflow induced by glucose/oxygen deprivation were significantly reduced in the absence of external Ca(2+) as well as by the addition of the mitochondrial Na(+)-Ca(2+) exchanger blocker, CGP 37157, and of the endoplasmic reticulum Ca(2+)/ATPase blocker, thapsigargin. +/-AP5, an NMDA receptor antagonist, and 5,7-diCl-kynurenic acid, an antagonist of the glycine site associated to NMDA receptor, markedly depressed glucose/oxygen deprivation-induced acetylcholine and glutamate overflow as well. Our results suggest that in vitro simulated ischaemia evokes acetylcholine and glutamate overflow from the guinea pig ileum, which is partly linked to an increase in intracellular Ca(2+) concentration dependent on both Ca(2+) influx from the extracellular space and Ca(2+) mobilization from the endoplasmic reticulum and mitochondrial stores. During glucose/oxygen deprivation, ionotropic glutamate receptors of the NMDA type exert both a positive feedback modulation of glutamate output and contribute to increased acetylcholine overflow.

Functional interaction between alpha2-adrenoceptors, mu- and kappa-opioid receptors in the guinea pig myenteric plexus: effect of chronic desipramine treatment.

The existence of a functional interplay between alpha(2)-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to alpha(2)-adrenoceptor, mu- and kappa-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of alpha(2)-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric mu- and kappa-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively alpha(2A)-adrenoceptor and kappa-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), mu-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory G(i/o) proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory G(i/o) proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the mu-opioid agonist might compensate for the development of alpha(2A)-adrenoceptor and kappa-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of alpha(2)-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for mu-opioid, but not for kappa-opioid receptors.

Role of glutamatergic neurotransmission in the enteric nervous system and brain-gut axis in health and disease.

Several studies have been carried out in the last 30 years in the attempt to clarify the possible role of glutamate as a neurotransmitter/neuromodulator in the gastrointestinal tract. Such effort has provided immunohistochemical, biomolecular and functional data suggesting that the entire glutamatergic neurotransmitter machinery is present in the complex circuitries of the enteric nervous system (ENS), which participates to the local coordination of gastrointestinal functions. Glutamate is also involved in the regulation of the brain-gut axis, a bi-directional connection pathway between the central nervous system (CNS) and the gut. The neurotransmitter contributes to convey information, via afferent fibers, from the gut to the brain, and to send appropriate signals, via efferent fibers, from the brain to control gut secretion and motility. In analogy with the CNS, an increasing number of studies suggest that dysregulation of the enteric glutamatergic neurotransmitter machinery may lead to gastrointestinal dysfunctions. On the whole, this research field has opened the possibility to find new potential targets for development of drugs for the treatment of gastrointestinal diseases. The present review analyzes the more recent literature on enteric glutamatergic neurotransmission both in physiological and pathological conditions, such as gastroesophageal reflux, gastric acid hypersecretory diseases, inflammatory bowel disease, irritable bowel syndrome and intestinal ischemia/reperfusion injury.

Interferon-gamma and interferon-beta affect endogenous catecholamines in human peripheral blood mononuclear cells: implications for multiple sclerosis.

Interferon (IFN)-gamma plays a pivotal role in the pathogenesis of multiple sclerosis (MS), while IFN-beta may be able to modify the clinical course of the disease, eventually also by counterbalancing IFN-gamma-mediated effects. Catecholamines (CA) exert important effects on the immune response, both as transmitters between the nervous and the immune system, as well as autocrine/paracrine mediators in immune cells, and several lines of evidence support their involvement in MS. In particular, dysregulated production of CA seems to occur in peripheral blood mononuclear cells (PBMCs) of MS patients. We assessed the effects of IFN-beta and IFN-gamma on endogenous CA in PBMCs. In cultured PBMCs stimulated with phytohaemagglutinin (PHA), IFN-beta increased CA production and induced CA release in the culture medium, while IFN-gamma decreased both CA production and the expression of mRNA for the CA-synthesizing enzyme tyrosine hydroxylase. Coincubation with both IFNs prevented the inhibitory effect of IFN-gamma, as well as the stimulatory effect of IFN-beta. IFNs are the first physiological compounds shown to affect endogenous CA in PBMCs: in view of the role of CA-dependent mechanisms in the immune response, these findings may help to better understand the mechanisms of action of IFN-beta as an immunomodulatory drug in MS.

Medical healthcare use in Parkinson's disease: survey in a cohort of ambulatory patients in Italy.

BACKGROUND: Parkinson's disease (PD) is a chronic neurodegenerative disease which at present has no cure, and it usually results in severe disability. The burden of PD increases as the illness progresses, resulting in the extensive utilisation of both health and community services. Knowledge of healthcare use patterns and of their determinants may greatly contribute to improve patient care, however few studies have examined this issue in PD. The present study was devised to describe the type of and reasons for medical healthcare resource use in persons with PD attending a Centre for PD and Movement Disorders, and to examine drug prescriptions issued on such occasions. METHODS: The study was a retrospective, cross-sectional survey in a cohort of ambulatory patients with PD, conducted by means of standard interviews. RESULTS: In the year before the study, 92 (70.8%) of 130 patients used medical healthcare resources: 1/5 of the patients was admitted to hospital, 1/5 to emergency room, 2/5 were visited by a non-neurology specialist, and 1/4 by the GP. Reasons were: nearly 20% programmed hospital admissions and visits, and more than 25% injuries and musculo-skeletal diseases. Other conditions typically occurring in PD (e.g. dementia, diabetes and cardio- and cerebro-vascular disease) were less frequently involved. On such occasions, drugs for PD were occasionally changed, however drug prescriptions for other indications were issued to more than 66% of the patients. CONCLUSION: Several physicians other than the neurologist may take care of PD patients on different occasions, thus emphasising the need for communication between the reference neurologist and other physicians who from time to time may visit the patient.

Interaction between NMDA glutamatergic and nitrergic enteric pathways during in vitro ischemia and reperfusion.

Nitric oxide (NO) and glutamate, via N-methyl-d-aspartate (NMDA) receptors, participate to changes in neuromuscular responses after ischemic/reperfusion (I/R) injury in the gut. In the present study we investigated the existence of a possible interplay between nitrergic and NMDA receptor pathways in the guinea pig ileum after in vitro I/R injury, resorting to functional and biomolecular approaches. In normal metabolic conditions NMDA concentration-dependently enhanced both glutamate (analyzed by high performance liquid chromatography with fluorimetric detection) and NO (spectrophotometrically quantified as NO2(-) and NO3(-)) spontaneous overflow from isolated ileal segments. Both effects were reduced by the NMDA antagonists, (-)-AP5 (10µM) and 5,7-diCl-kynurenic acid (10µM, 5,7-diCl-KYN). N(ω)-propyl-l-arginine (1µM, NPLA) and 1400W (10µM), respectively, nNOS and iNOS inhibitors, reduced NMDA-stimulated glutamate overflow. After in vitro I/R, glutamate overflow increased, and returned to control values in the presence of NPLA and 1400W. NO2(-) and NO3(-) levels transiently increased during I/R and were reduced by both (-)-AP5 and 5,7-diCl-KYN. In longitudinal muscle myenteric plexus preparations, iNOS mRNA and protein levels increased after in vitro I/R; both parameters were reduced to control values by (-)-AP5 and 5,7-diCl-KYN. Both antagonists were also able to reduce ischemia-induced enhancement of nNOS mRNA levels. Protein levels of GluN1, the ubiquitary subunit of NMDA receptors, increased after I/R and were reduced by both NPLA and 1400W. On the whole, this data suggests the existence of a cross-talk between NMDA receptor and nitrergic pathways in guinea pig ileum myenteric plexus, which may participate to neuronal rearrangements occurring during I/R.

Dopaminergic modulation of oxidative stress and apoptosis in human peripheral blood lymphocytes: evidence for a D1-like receptor-dependent protective effect.

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100-500 microM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1-5 microM decreased ROS levels and apoptosis. DA (both 1 and 500 microM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 microM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.

Stimulation with phytohaemagglutinin induces the synthesis of catecholamines in human peripheral blood mononuclear cells: role of protein kinase C and contribution of intracellular calcium.

Although it is now established that immunocompetent cells produce catecholamines (CA), which in turn may act as autocrine/paracrine mediators, little is known about the mechanisms regulating CA production in these cells. In the present study, evidence is provided that stimulation of human cultured peripheral blood mononuclear cells (PBMCs) with phytohaemagglutinin (PHA) induces the expression of tyrosine hydroxylase (TH) mRNA and subsequently increases intracellular CA levels through protein kinase C (PKC) activation and the contribution of intracellular Ca(++)-dependent mechanisms. Increased production of CA in PHA-stimulated PBMCs suggests a preferential involvement of catecholaminergic pathways in the functional modulation of activated cells. These findings may help to better define the role of immunocompetent cell-derived CA in the neuroimmune network.

Catecholamine production and tyrosine hydroxylase expression in peripheral blood mononuclear cells from multiple sclerosis patients: effect of cell stimulation and possible relevance for activation-induced apoptosis.

Sympathoadrenergic mechanisms may play a role in multiple sclerosis (MS). We examined catecholamine (CA) levels and production and tyrosine hydroxylase (TH) expression in peripheral blood mononuclear cells (PBMCs) from MS patients, and the correlation between CA production and apoptosis in PBMCs. PBMCs from MS patients had increased norepinephrine (NE) levels. However, phytohaemagglutinin (PHA)-stimulated PBMCs from MS patients with active disease synthesized less dopamine (DA) than cells from both healthy controls and patients with inactive disease. PBMCs from patients with inactive disease showed lower expression of TH. Pharmacological inhibition of TH in cultured PBMCs stimulated with PHA reduced the percentage of apoptotic cells. Since a failure of activation-induced apoptosis in immune cells may be involved in MS, it is suggested that altered CA production by PBMCs may be implicated in such dysregulation.

Dopaminergic D1-like receptor-dependent inhibition of tyrosine hydroxylase mRNA expression and catecholamine production in human lymphocytes.

Activation of human peripheral blood mononuclear cells (PBMC) triggers endogenous production of catecholamines (CA) through protein kinase (PK) C-dependent induction of tyrosine hydroxylase (TH; EC 1.14.16.2), the first and rate-limiting enzyme in the synthesis of CA. Since CA themselves are major mediators of the neural input to the immune system, we have examined their ability to affect PKC-induced TH mRNA expression and CA production in human isolated PBMC. In T- and B-lymphocytes (but not in monocytes) the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) (but not its inactive analogue 4alpha-phorbol-12,13-didecanoate) induced TH mRNA expression which was followed by an increase in the amount of intracellular CA. Coincubation of human PBMC with dopamine (DA) (but not with norepinephrine or epinephrine) inhibited TPA-induced TH mRNA expression. The effect of DA was concentration-dependent and was mimicked by the dopaminergic D1-like receptor agonist SKF-38393 but not by the D2-like receptor agonist bromocriptine. The D1-like antagonist SCH-23390 shifted to the right the concentration-response curves of both DA and SKF-38393, while neither the D2-like antagonist domperidone, nor the alpha1-adrenoceptor antagonist prazosin, the alpha2-adrenoceptor antagonist yohimbine, or the beta-adrenoceptor antagonist propranolol affected to any significant extent the inhibitory effect of DA. SKF-38393 also significantly reduced TPA-induced increase of intracellular CA, an effect which was antagonized by SCH-23390. It is thus suggested that in human T- and B-lymphocytes PKC activation leads to TH mRNA expression and subsequent increase of intracellular CA, which can be inhibited by D1-like receptor activation. Inhibition of intracellular CA production in human PBMC promotes cell survival through reduction of activation-induced apoptosis, and dopaminergic modulation of TH expression and intracellular CA content may thus represent a novel mechanism in the cross-talk between the nervous and the immune system as well as among immune system cells.

Dopaminergic modulation of apoptosis in human peripheral blood mononuclear cells: possible relevance for Parkinson's disease.

Dopamine (DA) modulates apoptosis in neuronal and non-neuronal cells, and dopaminergic pathways contribute to neurodegenerative disease. Human lymphocytes express dopaminergic receptors and DA transporters, and synthesize endogenous catecholamines, which may modulate apoptosis in these cells. In the present study, dopaminergic modulation of apoptosis was investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Twenty-four-hour DA reduced at 0.1-5 x 3 10(-6) M and enhanced at 1-5 x 310(-4) M spontaneous apoptosis. DA 1 x 310(-6) M was inhibited by the D1-like receptor antagonist SCH 23390 1 x 310(-6) M, but not by the D2-like receptor antagonists domperidone 1 x 3 10(-6) M or haloperidol 1 x 3 10(-6) M, while the effect of DA 5 x 3 10(-4) M was prevented by the antioxidants glutathione 5-10 mM or N-acetyl-l-cysteine 1-10 mM. Intracellular reactive oxygen species were respectively reduced and increased by 1-3 h incubation with DA 0.1-10 x 3 10(-6) M and 1-5x310(-4) M. Twenty-four-hour DA 1 x 3 10(-6) M or 5 x 3 10(-4) M had no effect on PBMC expression of Cu/Zn superoxide dismutase or Bcl-2; however, DA 5 x 3 10(-4) M decreased caspase-3 activity. In human PBMCs, DA seems to promote apoptosis through oxidative mechanisms but may also result in cell rescue from apoptotic death possibly through activation of D1-like receptors. The dual effect of DA on human PBMCs closely resembles that on striatal neurons. Lymphocytes of patients with Parkinson's disease may show reduced DA content and impaired DA transporter immunoreactivity. Human PBMCs may thus represent a simple and readily accessible model to study DA-related mechanisms relevant for neurodegenerative disease.

Evidence for a glutamatergic modulation of the cholinergic function in the human enteric nervous system via NMDA receptors.

Several reports suggest that enteric cholinergic neurons are subject to a tonic inhibitory modulation, whereas few studies are available concerning the role of facilitatory pathways. Glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), has recently been described as an excitatory neurotransmitter also in the guinea-pig enteric nervous system (ENS). The present study aimed at investigating the presence of glutamatergic neurons in the ENS of the human colon. At this level, the presence of ionotropic glutamate receptors of the NMDA type, and their possible interaction with the enteric cholinergic function was also studied. In the human colon, L-glutamate and NMDA concentration dependently enhance spontaneous endogenous acetylcholine overflow in Mg2+-free buffer, both effects being significantly reduced by the antagonists, (+/-)-2-amino-5-phosphonopentanoic acid (+/- AP5) and 5,7-diCl-kynurenic acid. In the presence of Mg2+, the facilitatory effect of L-glutamate changes to inhibition, while the effect of NMDA is significantly reduced. In addition, morphological investigations reveal that glutamate- and NR1-immunoreactivities are present in enteric cholinergic neurons and glial cells in both myenteric and submucosal plexus. These findings suggest that, as described for the guinea-pig ileum, glutamatergic neurons are present in enteric plexuses of the human colon. Modulation of the cholinergic function can be accomplished through NMDA receptors.

Sympathetic denervation-induced changes in G protein expression in enteric neurons of the guinea pig colon.

Chronic sympathetic denervation entails subsensitivity to alpha(2)-adrenoceptor agonists and supersensitivity to kappa- and mu-opioid receptor agonists modulating cholinergic neurons in the guinea pig colon. A possible role for signal transduction G proteins in contributing to development of these sensitivity changes was investigated. Pertussis toxin (PTX), a blocker of the G(i/o)-type family of G proteins significantly reduced the inhibitory effects of UK14,304 (alpha(2)-adrenoceptor agonist), U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on acetylcholine (ACh) overflow in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. In this experimental condition, immunoblot analysis revealed reduced levels of G(alphao), G(alphai2), G(alphai3) and G(beta) in myenteric plexus synaptosomes. On reverse, synaptosomal levels of G(alphai1) and G(alphaz), a PTX-insensitive G-protein, increased after chronic ablation of the sympathetic pathways. These data suggest that changes in the function and expression of inhibitory G proteins coupled to alpha(2)-adrenoceptors, kappa- and mu-opioid receptors occur in the myenteric plexus of the guinea pig colon after chronic sympathetic denervation. The possibility that regulation of G proteins represents one of the biochemical mechanisms at the basis of the changes in sensitivity of enteric cholinergic neurons to alpha(2)-adrenoceptor, kappa- and mu-opioid receptor agonists is discussed.

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus.

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Antagonism of ionotropic glutamate receptors attenuates chemical ischemia-induced injury in rat primary cultured myenteric ganglia.

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN(1) and GluA(1-3), GluK(1-3) respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA(1-3) subunits remained unchanged, while, the number of GluK(1-3)-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100-500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.